1. Field of the Invention
The present invention relates to calcitonin (CT). More particularly, the present invention relates to the expression of calcitonin in yeasts.
2. Description of the Related Arts
Calcitonin was identified as a hormone factor for reducing the concentration of calcium ion in serum in 1962. It was also noted that calcitonin is secreted by C-cells from the thyroid gland. Busolati G et al. (1967) proposed that calcitonin is composed of 32 amino acids, the first and the seventh cysteines have a disulfide bond, and proline at the C-terminal end is amidated. Sexton, P. M. et al. (1999) reported that the amidation of proline at the C-terminal end is critical for the bioactivity of calcitonin. In vitro study by Sexton, P. M. et al. (1983) and in vivo study by Chamber, T. J. et al. (1983) have demonstrated that the adenylyl cyclase and cAMP dependent protein kinase can be activated by the binding of calcitonin and calcitonin receptor on cell membrane to decrease osteoclast activity thereby alleviating osteoporosis. It is known that calcitonin anticipates calcium ion metabolism and inhibits osteoporosis resulting from osteoclast activity; therefore, calcitonin is considered an effective agent for the treatment of osteoporosis.
Gennari, C. et al. (1999) and Avioli, L. V. (1997) reported that calcitonin provides both a treatment and a preventive effect. Calcitonin can be used clinically to treat bone disorders, such as Paget's disease, osteoporosis, hypercalcemia malignancy. At present, the clinically used calcitonin is derived from human, salmon, porcine, and eel. Sexton, P. M. et al. (1999) reported that the bioactivity of calcitonin derived from salmon is especially high compared to other sources. The present clinically used calcitonin is manufactured through chemical synthesis, which is costly and limited by the length of amino acids. With the development of molecular technology in the last decade, a trend of protein drug production using living organisms (Ivanov, I. 1987; Ishikawa, H. 1996; 1999) has been arisen.
The present technology using recombinant proteins to produce calcitonin can be classified as Escherichia coli production, animal cell production, and yeast production. The advantages and disadvantages of the three productions are discussed in the following.
U.S. Pat. No. 6,210,925B2 to Unigene Laboratories, Inc. discloses a method for calcitonin production by E. coli having the advantage of high production; however, the formation of inclusion bodies in E. coli decreases the solubility of calcitonin.
Takahashi K. I. et al. (Peptides, 1997; 18(3):439–444) disclose a method for calcitonin production by nonendocrine animal cell lines, such as COS-7 and CHO. The last amino acid of the precursor of recombinant calcitonin is glycine which can be amidated easily and the product is identical to naturally occurring calcitonin without any chemical modification. The disadvantages of this method include low production rate and high cost.
Micronova R. et al. (FEMS Microbiology Letter, 1991; 67(1):23–28) disclose a method for human calcitonin production by Saccharomyces cerevisiae. The product can be secreted to the medium and any purification process is unnecessary. The production rate in yeast is between the rates of animal cells and E. coli, however, an additional C terminal amidation is necessary.
The above mentioned methods have several disadvantage, hence there is still a need for calcitonin production with high production rate and simple purification process.